Thirty million Americans have significant hearing problems that range in severity from modest difficulty with speech comprehension to profound deafness. Hearing impairment affects people of all ages. One child in a thousand is born deaf, and another one in a thousand becomes deaf before adulthood. A quarter of our population over 60 years of age is hearing-impaired, and half of those older than 80 are afflicted.
Most deaf individuals suffer from sensorineural hearing loss that results from damage to hair cells, the sensory receptors of the inner ear. Each cochlea normally contains about 16,000 hair cells, which convert mechanical inputs derived from sound into electrical signals that the brain can interpret. Similar cells in the vestibular labyrinth mediate responsiveness to acceleration and thus underpin our sense of balance. Human hair cells may be lost throughout life as a result of genetic conditions, infections, ototoxic drugs, acoustical trauma, and aging. Because these cells are not replaced by cellular division, their disappearance is associated with a gradual decline of our senses of hearing and equilibrium. In the hope of understanding normal hearing—as well as its development, its deterioration, and its possible restoration—our group is investigating the molecular structure and operation of hair cells from the vertebrate internal ear.
Coherent Motion of Stereocilia in the Hair Bundle of the Bullfrog's Sacculus
The hair bundle, the mechanoreceptive organelle of the hair cell, is highly sensitive because its transduction channels open over a very narrow range of displacements. The synchronous gating of transduction channels also underlies the active hair-bundle motility that amplifies and tunes responsiveness. The extent to which the gating of independent transduction channels is coordinated depends on how tightly individual stereocilia are constrained to move as a unit. We therefore wish to understand how signals propagate across a hair bundle to reach the full complement of channels.
A conclusive examination of the propagation of mechanical forces across a bundle requires simultaneous measurements of the positions of two stereocilia with a subnanometer spatial and a submillisecond temporal resolution, for these are the scales typical of stereociliary movements during hearing. Because interferometry can detect motions with the requisite precision, we constructed a dual-beam, differential laser interferometer and used it to examine the correlations between the thermal motions of individual stereocilia both in quiescent and in spontaneously oscillating hair bundles. We found that thermal movements of stereocilia located as far apart as the bundle's opposite edges display high coherence and zero phase lag over a wide range of frequencies. Because the mechanical degrees of freedom of stereocilia are strongly constrained, a force applied anywhere in the hair bundle deflects the structure as a unit. This feature assures the concerted gating of transduction channels that maximizes the sensitivity of mechanoelectrical transduction and enhances the hair bundle's capacity to amplify its inputs.
The Origin and Polarization of Regenerating Hair Cells in the Zebrafish's Lateral Line
In addition to displaying an axis of basolateral polarity, epithelial cells are often polarized perpendicularly to this axis, within the plane of the epithelium. The latter type of cellular organization, termed planar cell polarity, is exemplified by the orientation of the hair bundle that projects from the apical surface of each sensory hair cell. Because the hair bundle's axis of morphological polarization defines the cell's axis of responsiveness to mechanical stimuli, the senses of hearing and equilibrium rely on the coordinated orientation of hair cells across the sensory epithelium.
The restoration of planar cell polarity is an essential but poorly understood step toward physiological recovery during sensory-organ regeneration. We investigated this issue in the lateral line of the zebrafish by observing the production of new hair cells after lesioning with an aminoglycoside antibiotic. We identified a molecular marker that permitted us to recognize for the first time a population of transient hair-cell progenitors that are produced by putative stem cells. Within each neuromast, hair cells regenerate in pairs along a single axis established by the restricted localization and oriented division of their progenitors. By analyzing mutants lacking the planar-polarity determinant Vangl2, we ascertained that the uniaxial production of hair cells and the subsequent orientation of their hair bundles are controlled by distinct pathways, whose combination underlies the establishment of hair-cell orientation during development and regeneration. This mechanism may represent a general principle governing the long-term maintenance of planar cell polarity in remodeling epithelia.
Specificity of Afferent Synapses onto Plane-Polarized Hair Cells
Although we know several of the signals that direct growing axons to the appropriate part of the nervous system, we lack information about the factors involved in the selection of specific synaptic targets. The lateral-line system constitutes a promising experimental system for exploration of this issue, for the choice of targets is binary: each hair cell is sensitive to stimulation either in the rostral or in the caudal direction; an afferent neuron must select targets of one polarity or the other, or conceivably both. Moreover, because the alternative targets are both hair cells—and the daughters of a single mitotic division—there are likely to be minimal differences between them that guide selective innervation.
We have explored this issue in the developing zebrafish. By injecting DNA constructs encoding the membrane-bound red-fluorescent protein mCherry under the control of the islet1 promoter, we labeled individual afferent neurons in their entirety. We then used confocal microscopy to examine the relation of each axonal terminal to hair cells of the two polarities. The results were highly consistent: at least 95 percent of the terminals formed by a given fiber occurred on hair cells of a single, common orientation. Electron microscopy confirmed that the contacts were mature synapses endowed with ribbons and clustered vesicles. When a fiber contacted two or more neuromasts, it consistently synapsed with hair cells of the same orientation in each. Moreover, when the hair cells were eliminated by Cu2+ treatment, a fiber always innervated regenerated hair cells of the polarity chosen originally. These results establish the lateral-line system as a simple model for the investigation of synaptic-target selection and question whether synapse formation depends on chemical labels or electrical activity.
